Project Summary Cyclic AMP is a universal second messenger and is essential for the chronotropic, inotropic and lusitropic effects during the `fight-or-flight' response. Dysregulation of cAMP signaling induces hypertrophic growth and ventricular dysfunction, eventually leading to heart failure. Adenylyl cyclase (AC) production of cAMP is controlled on many levels, including the formation of macromolecular complexes that generate localized pools of cAMP. Disorganization of cAMP compartmentation and a loss of hormonal specificity occur in models of chronic heart failure, suggesting a mechanism for the cardiotoxic effects of the cAMP pathway. We have previously identified multiple cardiac A-kinase anchoring proteins (AKAPs) that scaffold AC near upstream and downstream signaling partners to provide a means of spatial and temporal regulation of cAMP and cardiac function. But is AKAP scaffolding required for all cAMP effectors? We hypothesize that 1) AKAP-independent anchoring of AC to effector complexes is a major contributor to cAMP sensitivity and 2) both AKAP-dependent and AKAP-independent macromolecular AC-effector complexes are required for cardiac function. In support of this hypothesis, we have identified a new AC scaffolding protein, the Popeye Domain-Containing protein, Popdc. Popdc isoforms are transmembrane proteins that are important for pacemaker function, reperfusion injury, and muscle regeneration. Importantly, Popdc represents a novel cAMP effector, with a cytosolic cAMP binding domain that regulates protein-protein interactions. Clinically, mutation of human Popdc1 (S201F) reduces cAMP binding by 50% and causes Limb-Girdle Muscular Dystrophy and Cardiac Arrhythmia. In sinoatrial node, cAMP-dependent Popdc regulation of the potassium channel TREK-1 acts to control heart rate. Our recent studies of AC9-/- mice show that AC9 is also an important regulator of basal cardiac function and heart rate, while preliminary data show a complex of AC9-Popdc-TREK. Additionally, Popdc regulates AC9 activity in a cAMP-dependent manner. Thus, the Popdc proteins have all the scaffolding, feedback, and effector regulatory properties that AKAP-PKA complexes create, but bundled in one unique protein. Three specific aims are proposed to examine the role of this novel AC complex on cardiac function. Aim 1, Identify specificity and interaction sites for AC-Popdc complexes. Aim 2, Determine the mechanism of Popdc regulation of AC activity. Aim 3, Examine functional roles for AC-Popdc complexes in heart. Our studies have important implications not only for cardiac muscle, but for skeletal muscle regulation and possibly additional AC isoforms.